Carotenoid (also called “carotinoid”) is a general term for pigments occurring abundantly in nature, which are built up from an isoprene backbone of 40 carbon atoms. To date, more than 700 species of carotenoids have been isolated (Britton, G., Liaaen-Jensen, S., and Pfander, H., Carotenoids Handbook, Birkhauser Verlag, Basel, 2004). Recently, prophylactic effects of carotenoids on chronic diseases such as cancer have attracted people's attention, and a great number of reports have been made (see, for example, H. Nishino, M. Murakoshi and M. Yano, Food Style 21, 4, 53-55, 2000; Nishino, H. et al, “Carotenoids in cancer chemoprevention”, Cancer Metastasis Rev. 21, 257-264, 2002; Mayne, S. T., “β-Carotene, carotenoids, and disease prevention in humans”, FASEB J., 10, 690-701, 1996).
Although carotenoids have a huge variety of species, species which have been used in prophylactic studies (human epidemiological/clinical tests, animal administration tests, etc.) are extremely limited. Those carotenoids include β-carotene (also called β-carotine; chemically synthesized product), lycopene (also called lycopine; extracted from tomato), α-carotene (also called α-carotine; extracted from palm oil), lutein (extracted from marigold), astaxanthin (extracted from krill or Haematococcus alga, or chemically synthesized), fucoxanthin (extracted from edible marine algae) and β-cryptoxanthin (extracted from mandarin orange). The results of cancer prophylactic studies using these pigments gradually revealed that cancer prophylactic effects of carotenoids vary depending on the species of carotenoids. As an example, the results of experiments using mice conducted by Nobuo Takasuka et al. of the Research Institute, National Cancer Center (Report of 1996 Meeting on Carotenoid Research) will be shown below. The incidence of lung cancer (ddy mouse lung two-stage carcinogenesis model) was 40% in α-carotene-administered mice, 70% in lutein- or astaxanthin-administered mice and 139% in β-carotene-administered mice, when the incidence in control mice without carotenoid administration was taken as 100%. The incidence of liver cancer (mouse spontaneous liver carcinogenesis model) was 30% in astaxanthin- or fucoxanthin-administered mice, 50% in α-carotene- or lutein-administered mice, 70% in β-carotene-administered mice, and 100% in lycopene-administered mice, when the incidence in control mice without carotenoid administration was taken as 100%. The incidence of skin cancer (mouse skin carcinogenesis model) was 10% in fucoxanthin- or lycopene-administered mice, and 100% in astaxanthin-administered mice, when the incidence in control mice without carotenoid administration was taken as 100%. Comparison of the results on these three carcinogenesis models reveals that lycopene which was highly effective in inhibiting lung cancer and skin cancer is not effective in inhibiting liver cancer; and that astaxanthin which was highly effective in inhibiting liver cancer is not effective in inhibiting skin cancer. Further, as a result of epidemiological tests and clinical tests, it has been reported that, among dietary carotenoids, only lycopene was confirmed as a prophylactic carotenoid against prostate cancer (see Giovannucci, E., Ascherio, A., Rimm, E. B., Stampfer, M. J., Colditz, G. A., Willet, W. C., “Intake of carotenids and retinol in relation to risk of prostate cancer”, J. National Cancer Institute 87, 1767-1776, 1995; Vogt, T. M. et al, “Serum lycopene, other serum carotenoids, and risk of prostate cancer in US Blacks and Whites”, Am. J. Epidemiol. 155, 1023-1032, 2002). Recently, high prophylactic effect of β-cryptoxanthin on lung cancer has been gradually elucidated (see Yuan, J. M., Stram, D. O., Arakawa, K., Lee, H. P. and Yu M. C., “Dietary cryptoxanthin and reduced risk of lung cancer: the Singapore Chinese Health Study”, Cancer Epidemiol. Biomarkers Prev. 12, 890-898, 2003; Mannisto, S. et al, “Dietary carotenoids and risk of lung cancer in a pooled analysis of seven cohort studies”, Cancer Epidemiol. Biomarkers Prev. 13, 40-48, 2004). In addition to cancer prophylactic effects, it has been also reported that carotenoids are very likely to be effective in preventing chronic diseases in the cardiovascular system, chronic diseases in the eye such as cataract, and chronic diseases such as osteoporosis. For example, it has been reported that carotenoids which are expected to be effective on chronic diseases in the eye (age-related macular degeneration, cataract, etc.) are lutein and zeaxanthin alone among dietary carotenoids (see Semba, R. D. and Dagnelie, G., “Are lutein and zeaxanthin conditionally essential nutrients for eye health?”, Med. Hypotheses 61, 465-472, 2003; Mazaffarieh, M., Sacu, S. and Wedrich, A., “The role of the carotenoids, lutein and zeaxanthin, in protecting against age-related macular degeneration: A review based on controversial evidence”, Nutr. J., 2, 20, 2003).
The results described so far indicate that only about 10 out of 700 or more species of carotenoids have been examined for their prophylactic effects on chronic diseases such as cancer in studies actually using animal individuals, and that each carotenoid has characteristic individuality in prophylactic effect on chronic diseases such as cancer. It is believed that the major reason why the number of species of carotenoids actually examined is so mall is because those carotenoids which can be extracted, purified or chemically synthesized in large quantities are limited to the above-mentioned carotenoids.
As a promising means to solve the above problem, a method may be considered in which a carotenoid of interest is mass-produced in carotenoid biosynthesis gene-transferred yeast or Escherichia coli. For example, Shimada et al. of Kirin Brewery introduced a carotenoid biosynthesis gene cluster into a food yeast Candida utilis which naturally does not biosynthesize carotenoids, expressed the genes and succeeded in synthesizing 7.8 mg/g of lycopene (dry weight) (Shimada, H., Kondo, K., Fraser, P. D., Miura, Y., Saito, T., and Misawa, N., “Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway”, Appl. Environ. Microbiol., 64, 2676-2680, 1998). According to the gene recombination technique, it becomes possible to produce such carotenoids that have not been found in nature or found only in trace amounts, by a combination of various biosynthesis genes. For example, Takaichi et al. of Nippon Medical School have succeeded in producing parasiloxanthin (which is only reported to be found in catfish in a trace amount) as a major carotenoid product in a recombinant E. coli (Takaichi, S., Sandmann, G., Schnurr, G., Satomi, Y., Suzuki, A., and Misawa, N. “The carotenoid 7,8-dihydro-Ψ end group can be cyclized by the lycopene cyclases from the bacterium Erwinia uredovora and the higher plant Capsicum annuum”, Eur. J. Biochem., 241, 291-296, 1996). There is another report that astaxanthin-β-diglucoside, a “non-natural type” carotenoid not found in nature, was produced in a recombinant E. coli (Yokoyama, A., Shizuri, Y.,and Misawa, N., Production of new carotenoids, astaxanthin glucosides, by Escherichia coli transformants carrying carotenoid biosynthetic genes. Tetrahed. Lett., 39, 3709-3712, 1998).
The carotenoid biosynthesis genes most commonly used in the preparation of recombinant microorganisms for various carotenoid productions are derived from Erwinia bacteria (such as Erwinia uredovora; recently, this bacterium is called Pantoea ananatis). Six genes have been isolated from Erwinia bacteria; they are crtE, crtB, crtI, crtY, crtZ and crtX. The functions of the biosynthesis enzymes encoded by these genes (CrtE, CrtB CrtI, CrtY, CrtZ and CrtX) are shown in FIG. 1 (see Non-Patent Document 1). When biosynthesis of astaxanthin is intended, crtW gene derived from marine bacteria Paracoccus [such as Paracoccus sp. MBIC 01143 (Agrobacterium aurantiacum)] is additionally required (FIG. 1). Five gene have been isolated from Paracoccus bacteria; they are crtB, crtI, crtY, crtZ and crtW (see Non-Patent Document 1). The functions of crtB, crtI, crtY and crtZ genes are common in both bacteria. When Erwinia- or Paracoccus-derived crtE, crtB, crtI and crtY genes have been introduced and expressed in E. coli, the E. coli biosynthesizes β-carotene. When marine bacterium-derived crtW gene and Erwinia- or Paracoccus-derived crtZ gene are further introduced and expressed in the above E. coli, the recombinant E. coli begins to synthesize astaxanthin. Further, when Erwinia-derived crtX gene is introduced and expressed in this E. coli synthesizing astaxanthin, the recombinant E. coli begins to synthesize a “non-natural type” carotenoid, astaxanthin-β-diglucoside (FIG. 1).
As described so far, it is being demonstrated that carotenoid biosynthesis genes can be used to produce “rare” carotenoids which occur only in trace amounts in nature or “non-natural type” carotenoids existence of which has not been confirmed. On the other hand, the carotenoid biosynthesis genes which may be used for this purpose are limited to 25 genes. They are crtM (dehydrosqualene synthase), crtE (gps, al-3) (geranylgeranyl pyrophosphate synthase), crtB (psy, al-2) (phytoene synthase), crtN (dehydrosqualene desaturase), crtP (pdsl) (phytoene desaturase: addition of two double bonds), crtQ (zds) (ζ-carotene desaturase: addition of two double bonds), crtI (derived from Rhodobacter) (phytoene desaturase: addition of three double bonds and cis-trans isomerization), crtI (phytoene desaturase: addition of four double bonds and cis-trans isomerization), al-1 (phytoene desaturase: addition of five double bonds and cis-trans isomerization), crtY (crtL-P) (lycopene β-cyclase), crtL-c (lycopene ε-cyclase), crtYm (lycopene β-monocyclase), crtU (β-carotene desaturase), crtZ (β-carotene hydroxylase; β-C3-hydroxylase), crtW (bkt) (β-carotene ketolase; β-C4-oxygenase), crtO (derived from Synechocystis sp. PCC6803) (β-carotene monoketolase), crtX (zeaxanthin glucosyltransferase), crtC (hydroxyneurosporene synthase), crtD (methoxyneurosporene desaturase), crtF (hydroxyneurosporene o-methyltransferase), crtA (spheroidene monooxygenase), crtEb (lycopene elongase), crtYe/Yf (decaprenoxanthin synthase), zepl (zeaxanthin epoxydase) and ccs (capsanthin/capsorubin synthase) (see Lee, P. C. and Schmidt-Dannert, C., “Metabolic engineering towards biotechnological production of carotenoids in microorganisms”, Appl. Microbiol. Biotechnol. 60, 1-11, 2002; Teramoto, M., Takaichi, S., Inomata, Y., Ikenaga, H. and Misawa, N. “Structural and functional analysis of a lycopene β-monocyclase gene isolated from a unique marine bacterium that produces myxol”, FEBS Lett. 545, 120-126, 2003). In order to allow microorganisms such as E. coli to produce a wide variety of carotenoids, novel carotenoid biosynthesis genes must be isolated. However, cloning of novel carotenoid biosynthesis genes makes very slow progress. For example, while carotenoids occurring most abundantly in nature are those with β-ionone rings (in FIG. 1, β-carotene, zeaxanthin, canthaxanthin, astaxanthin, etc.), only two genes of enzymes which hydroxylate or oxygenate β-ionone rings have been isolated. They are genes encoding β-ionone ring-3-hydroxylase (β-C3-hydroxylase) (CrtZ) and β-ionone ring-4-ketolase (β-C4-ketolase; β-C4-oxygenase) (CrtW), respectively. These enzyme genes were isolated as early as in 1990 for crtZ and in 1995 for crtW, and analyzed for their functions. It is believed that a gene of β-ionone ring-2-hydroxylase is necessary for synthesizing carotenoids such as nostoxanthin in which the position 2 carbon of the β-ionone ring is hydroxylated. However, though there are some microorganisms producing such carotenoids (see Non-Patent Document 2), nothing has been found to date about such an enzyme or gene. It seems that the reason why the cloning of novel carotenoid biosynthesis genes is difficult is because those carotenoid biosynthesis genes obtainable by expression cloning in E. coli or by cloning using homology to existing carotenoid genes have already been obtained and all the remaining genes are not obtainable by these cloning methods.
Carotenoids consisting of carbon and hydrogen alone are called carotene, and carotenoids comprising oxygen-containing functional groups, such as hydroxyl group or keto group, in addition to carbon and hydrogen are called xanthophyll. Carotene and xanthophyll are greatly different in physical property and considerably different in physiological activity in the living body. For example, β-cryptoxanthin is a carotenoid in which one hydroxyl group is introduced at the position 3 carbon of β-carotene. It is known that the intake ratio of β-cryptoxanthin into the living body is 10 times higher than that ratio of β-carotene. β-Cryptoxanthin is a carotenoid which has been especially attracting attention in Japan recently. Data showing its prophylactic effects on large bowel cancer, cervix cancer, esophageal cancer, prostate cancer, rheumatoid and osteoporosis in addition to the above-described lung cancer are being gathered (Yano, M., Report of 2003 Meeting on Carotenoid Research and the above-mentioned Yuan, J. M., Stram, D. O., Arakawa, K., Lee, H. P. and Yu M. C., Cancer Epidemiol. Biomarkers Prev. 12, 890-898, 2003 and Mannisto, S. et al, Cancer Epidemiol. Biomarkers Prev. 13, 40-48, 2004). Such effects are not recognized in β-carotene. A carotenoid in which two hydroxyl groups are introduced at both positions 3 and 3′ of β-carotene is zeaxanthin (see FIG. 1). As described above partially, it is known that the physiological activity of zeaxanthin is different from that of β-cryptoxanthin. A carotenoid in which the both methylene groups at positions 4 and 4′ of zeaxanthin are converted to keto groups is astaxanthin (see FIG. 1). As described above partially, the physiological activity of astaxanthin in cancer prevention is also greatly different from that of β-carotene. On the other hand, a carotenoid in which two hydroxyl groups are further introduced at both positions 2 and 2′ of zeaxanthin is nostoxanthin. Generally, carotenoids such as nostoxanthin in which two hydroxyl groups are introduced at positions 2 and 2′ of β-ionone rings occur only in trace amounts in nature, and it is impossible to produce them in large quantities. Thus, prophylactic studies against various chronic diseases such as cancer cannot be conducted. The enzyme that introduces hydroxyl groups at positions 3 and 3′ of β-ionone rings is CrtZ. However, no CrtZ proteins having a 49% or less identity with the Erwinia uredovora-derived CrtZ reported in a paper in 1990 for the first time (Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K., J. Bacteriol. 172, 6704-6712, 1990) have been isolated to date. Among the CrtZ proteins which have been confirmed to have the same function, the enzyme that has the highest homology to the Erwinia uredovora-derived CrtZ is a Paracoccus zeaxanthinifaciens (old designation: Flavobacterium sp. R1534)-derived CrtZ (Pasamontes, L., Hug, D., Tessier, M., Hohmann, H. P., Schierle, J., and van Loon, A. P., Gene 185, 35-41, 1997) with a 50% identity.
(Non-Patent Document 1) Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T., Ohtani, T., and Miki, W., “Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level”, J. Bacteriol., 177, 6575-6584, 1995)
(Non-Patent Document 2) Yokoyama, A., Miki, W., Izumida, H., and Shizuri, Y., “New trihydroxy-keto-carotenoids isolated from an astaxanthin-producing marine bacterium”, Biosci. Biotech. Bioche., 60, 200-203, 1996)